Break-induced replication (BIR), also called recombination-dependent DNA replication, plays critically important roles both during DNA replication and in the maintenance of telomeres lacking telomerase. BIR between homologous chromosomes leads to loss of heterozyosity (LOH). In addition, BIR is important in the creation of chromosome rearrangements between ectopic homologous sequences, including deletions, nonreciprocal translocations and copy number variation. Studies are proposed to continue our analysis of the detailed molecular mechanisms of BIR and to characterize the properties of a repair replication fork. The first Aim uses an HO endonuclease-induced double-strand break (DSB) to initiate ectopic BIR. Experiments examine key proteins in that have distinct roles in BIR different from their roles in normal DNA replication, especially the Cdc7 kinase, the nonessential Pold subunit, Pol32, and PCNA. A novel PCNA allele that prevents BIR but not replication or gene conversion will be characterized. New mutations in both Pol32 and PCNA that affect BIR will be sought to localize the domains and protein interaction partners that are affected. Whether Pol32 is required whenever Pold is indispensible for DNA repair will also be examined. A second Aim focuses attention on BIR that arises specifically in the context of normal DNA replication. It has now become possible to induce site-specific nicks in G1 cells that will be converted to DSBs by replication. Three different approaches will be used: (1) a modified site-specific nuclease, Ani1- K277M on one strand, (2)a Flp-H305L enzyme that leaves a nick with a 3'-covalently bound Flp protein on one strand, and (3) a bacterial RepC protein that cleaves a 32-bp site to produce a nick with a 5'-attached protein. These systems make it possible for the first time to analyze in real time process of recombination-dependent replication re-start at a broken replication fork. Sister-chromatid repair and unequal sister chromatid exchange will be tested, genetically and by real-time DNA analysis. The third Aim turns to a yeast model of the mechanism of generating copy number variation as seen in many cancers now called MM-BIR, but whose details have not been analyzed. A DSB-induced system will examine intra- and inter-chromosomal template switches either between highly diverged homeologous sequences or where there is no homology at all, in both cases to create a selectable gene that allows us to obtain even multiple template switches.